Telomere has a structure composed of several proteins and DNAs located at both ends of chromosomes of eucaryotic organisms. Studies carried out hitherto have revealed that the telomere structure protects the chromosomes from a kind of DNA degradative enzyme, inadequate DNA repair, and the like. Furthermore, since the telomeric DNA is shortened with every cell division, it has been believed to play an important role in events referred to as cell aging and immortalization.
On the other hand, telomere is proven to have several features in view of structural aspects. More specifically, in almost all parts of chromosomal DNAs, adenine (hereinafter, referred to as A) base with thymine (hereinafter, referred to as T) base, and guanine (hereinafter, referred to as G) base with cytosine (hereinafter, referred to as C) base specifically form Watson-Crick type base pairs via a hydrogen bond, respectively. A double helix structure is constructed between double DNA strands that are complementary to one another, through causing a π-π stacking interaction among their base pairs (FIG. 1).
However, in telomeric DNA, almost all parts are composed of double strand DNAs that are complementary to one another, whereas the 3′ end is overhung and forming a single strand DNA. Although the length of the overhang part and the full length of telomere may vary depending on the species, the overhang has a length of approximately 50-100 bases, while the full length has about 10,000 base pairs at the initial stage in the case of humans. Moreover, other structural feature of the telomere sequence is that the strand having the overhang includes repeating sequences that are rich in G, and thus another stand which is complementary thereto includes repeating sequences that are rich in C.
Specifically, in the case of humans, the strand including the overhang has repeating 5′-TTAGGG-3′ (SEQ ID NO: 1), i.e., a G-rich sequence, and another has repeating 5′-CCCTAA-3′ (antisense strand of SEQ ID NO: 1), i.e., a C-rich sequence.
In recent years, the G-rich sequence in the telomere site has been particularly extensively studied on the ground that it has been revealed that this sequence can form a quadruple DNA structure referred to as “G-quadruplex”. As the G-quadruplex (guanine quadruplex structure), multiple patterns have been known such as: one referred to as a parallel type in which all four DNA strands are oriented to the same direction of from 5′ to 3′; or one referred to as an antiparallel type in which two strands are oriented to the same direction while the other two are oriented to the opposite direction, any of which having the features as shown in FIG. 2. In other words, four G bases form a structure referred to as “G quartet” via a Hoogsteen hydrogen bond, and the structure is maintained by a π-π stacking interaction of the G quartet faces.
Moreover, formation of the G-quadruplex structure requires coordination of the metal ions between a G-quartet face and a G-quartet face, and coordination of a K ion, Na ion and the like has been known. Details of formation of such a G-quadruplex in a telomere have been unclear for the present on suitable conditions as well as on the function performed thereby. However, it has been considered that the G-quadruplex would be an important structure that may relate to protection of the chromosome, aging of the cell, and the like.
Still further, it has also been elucidated that the G-rich sequence as described above is present not only in telomeres. For example, also in c-myc which is a carcinogenic gene, are present G-rich sequences that form G-quadruplexes. In addition, other genes such as c-kit, bcl-2, VEGF, H-ras and N-ras gene are found to have G-rich sequences that form a G-quadruplex in the promoter region. These are all genes that relate to malignant transformation and the like of cells, suggesting that the G-quadruplex may play an important role in human bodies. For the present, it is expected that there further exist G-rich sequences being capable of forming G-quadruplexes in the number of over three hundred thousand (hereinafter, such sequence may be referred to as a putative G-quadruplex sequence). There has been increasing interest in whether or not the G-quadruplex is formed therefrom, and the function performed by the structure when it is formed.
One of the most necessary techniques in such technical background is a method for specifically detecting a G-quadruplex. That is, there exists a need for a technique that enables the presence or absence of a G-quadruplex in a DNA in a sample, or the presence or absence of possibility of practical formation of a G-quadruplex from a DNA constituted with the putative G-quadruplex sequence in the sample to be determined. For example, in an attempt to determine the former by way of a prior art, the following may be illustrated. That is, a sample solution including a target DNA is provided, and a CD (Circular Dichroism) spectrum of this solution is measured. Then, an analysis may be made as to whether or not thus obtained CD spectrum is peculiar to the G-quadruplex. However, apparatuses for measuring the CD spectrum are very expensive and of large size. In addition, a long period of time of approximately 30 min is required for the analysis, and the number of the samples which can be analyzed in one trial is usually one; therefore, this technique is significantly inferior in terms of high throughput performance. Further, depending on the shape of the quadruplex, there may be a problem of failure in distinguishing the DNA from those having other forms by using CD.
As a means for resolving such a problem, a method may be referred to in which a probe material that generates a signal (absorbance, fluorescence intensity, etc.), which can be analyzed with an inexpensive apparatus depending on the presence or absence of a G-quadruplex, is mixed in the solution beforehand, and the signal in this solution is detected, as shown in FIG. 3. In such a method, specificity for the G-quadruplex of this probe would be of importance. More specifically, since a genomic DNA includes therein both a double stranded DNA structure that accounts for a large proportion of the same, and a single stranded DNA structure typified by the aforementioned overhang of a telomere, specificity for the G-quadruplex over these structures is important in comparison to these structures. The reason for this importance is that irrespective of the contemplation for determining the presence or absence of only the G-quadruplex, accurate detection of a G-quadruplex in a solution would be difficult when a signal is generated upon interaction of the probe also with any of the single stranded and double stranded DNAs, as a result of low specificity for the G-quadruplex of the probe.
In addition, when a prior art is used for examining a DNA of the putative G-quadruplex sequence in a sample with respect to formation of a G-quadruplex in effect, the process as shown in FIG. 4 would be employed. More specifically, a DNA having the putative G-quadruplex sequence is provided in a solution. Thereafter, this solution is maintained under a condition for permitting a G-quadruplex forming reaction, thereby allowing a G-quadruplex-forming reaction to be carried out. Then, the analysis on the presence or absence of a G-quadruplex in the solution following the reaction may be performed by measuring a CD spectrum. However, there can be accompanied by problems of the apparatus being expensive and of large scale, and in terms of high throughput, as described above also in this process. Thus, a probe for G-quadruplex detection as shown in FIG. 3 would be very useful if available, also in this case.
Specifically, this probe is mixed with the solution after the G-quadruplex-forming reaction described above, and the signal generated as a result may be measured, as shown in FIG. 5. In this process, since the DNA of the putative G-quadruplex sequence before the G-quadruplex-forming reaction can be any of single stranded and double stranded DNAs as described above, specificity of the probe for the G-quadruplex is extremely important. That is, accurate detection of the formed G-quadruplex becomes difficult when the aforementioned probe interacts also with the original structure (single strand or double stranded DNA) of the DNA of the putative G-quadruplex sequence to generate a signal, in the case in which the DNA of the putative G-quadruplex sequence did not form a G-quadruplex at all after the G-quadruplex-forming reaction, or in the case in which such formation was only partially executed. To the contrary, when the specificity of the probe for a G-quadruplex is extremely high, mixing of the probe in the solution prior to the G-quadruplex-forming reaction is permitted, and complication of the operation can be also obviated in the method shown in FIG. 5.
As described in the foregoing, the G-rich sequence that forms a G-quadruplex has been found in sites relating to cancer, aging or the like in chromosomes. Therefore, it is expected that disease diagnoses can be realized in future by identifying a G-quadruplex in a chromosome of a patient. Accordingly, development of a method for specifically detecting a G-quadruplex can be deemed as a very important problem. In addition, as characteristic features of the probe, it would be more advantageous of course, when the probe not only can detect a G-quadruplex or a DNA of the putative G-quadruplex sequence specifically but can yield a change such as increase or decrease of the signal depending on the amount of them being present, since not only mere a detection but also quantitative analysis is enabled.
Additionally, an enzyme referred to as telomerase has been known. This enzyme is a complex composed of an RNA and reverse transcriptase etc., whereby the repeating sequence in the G-rich strand at the telomere site which is shortened upon cell division can be replicated and thus extension is permitted (i.e., in the case of human, telomerase adds a 5′-TTAGGG-3′ sequence (SEQ ID NO: 1); hereinafter, this addition reaction being referred to as telomerase reaction). This enzyme is not usually found in human common somatic cells, but it is known to be expressed in a large amount in germ cells as well as most cancer cells. Thus, the occurrence or absence of malignant transformation of a subject cell can be examined using the telomerase activity as a marker.
As a conventional method for this purpose, a process referred to as TRAP assay has been known. The TRAP assay is explained below with reference to FIG. 6. In the TRAP assay, a solution containing a synthetic single stranded DNA provided as a template for telomerase is prepared first. This single stranded DNA usually consists of a sequence of 5′-AATCCGTCGAGCAGAGTT-3′ (SEQ ID NO: 2), which is referred to as TS primer. Accordingly, telomerase achieves addition of the 5′-TTAGGG-3′ sequence (SEQ ID NO: 1) to the 3′ end of this TS primer (see, NPL 3). It should be noted that this TS primer is not limited to this sequence, but any single stranded DNA that serves as a template of telomerase is acceptable.
Next, into this solution is added an extract from a cell sample or a tissue sample to which malignant transformation is suspected. After mixing, the solution is maintained under a condition in which a telomerase reaction can be permitted. In this step, provided that the sample is subject to malignant transformation, the telomerase reaction is caused from the TS primer as an origin since there exists a telomerase activity. To the contrary, the telomerase reaction is not caused without malignant transformation. Then, the DNA elongated by the telomerase reaction is finally amplified by PCR, and thus amplified DNA fragment is detected by an electrophoretic technique.
Since the lengths of DNAs obtained by the telomerase reaction vary widely, the obtained electrophoretic image may be as shown in FIG. 7. More specifically, when the used sample has a potent telomerase activity, the obtained DNA fragments appear as ladder bands representing the lengths within a broad range from short to long, with the color density of the bands more likely to be higher. To the contrary, since a long DNA fragment cannot be obtained from telomerase having a weak activity, ladder bands appear representing the lengths in a short range, with the color density of the bands more likely to be lower. Therefore, for identifying the malignant transformation of the sample, the range of the lengths, and the color density of the band of the DNA fragments on the electrophoretic image obtained as a result of the TRAP assay may be analyzed. However, quantitative analyses of these have been still difficult; therefore, merely a qualitative evaluation is attainable.
In these respects, when the probe for specific detection of a G-quadruplex illustrated in FIG. 3 and FIG. 5 is not only merely specific for the G-quadruplex, but can alter its signal intensity depending on the amount of the G-quadruplex being present, such a probe can be useful also in diagnoses of cancer using the telomerase activity as a marker. The flowchart of such a diagnosis is shown in FIG. 8.
Also in this method, the following steps are carried out in a similar manner to those in the operation shown in FIG. 6: preparing a solution containing a TS primer first; adding an extract from a cell sample or a tissue sample into this solution next, followed by mixing; then subjecting this solution to a telomerase reaction, and thereafter amplifying with a PCR method the DNA elongated by the telomerase reaction. However, in the method shown in FIG. 8, the solution after the PCR is not subjected to electrophoresis, but is subjected to a G-quadruplex-forming reaction similarly to the case shown in FIG. 5. Consequently, thus formed G-quadruplex is detected using a probe for G-quadruplex detection. The amount of the formed G-quadruplex reflects the intensity of the telomerase activity in this step; therefore, this method enables quantitative analysis of the telomerase activity and identification of malignant transformation, provided that the probe can alter its signal intensity depending on the amount of the G-quadruplex.
Needless to say, specificity of the probe for a G-quadruplex is also extremely important in such a method. That is, when the cell sample is not subject to malignant transformation, the telomerase reaction does not proceed. As a result, the TS primer being a single stranded DNA remains unchanged in the reaction liquid. Furthermore, the following PCR reaction does not also proceed, and thus the primer for the PCR reaction remains in the state of the single stranded DNA. Therefore, when the cell sample is not subject to malignant transformation, a large amount of single stranded DNAs will be finally present in the reaction mixture, whereby quantitative analyses of the telomerase activity and diagnoses of cancer may be difficult due to a signal generated from the probe via a reaction with these single stranded DNAs.
As explained in the foregoing, a method that enables specific detection of a G-quadruplex is very useful even in a state in which any one of single stranded and double stranded DNAs can be present in the solution. Moreover, when a quantitative performance is available, problems in conventional TRAP assays can be solved, in addition to enabling quantitative detection of a G-quadruplex. Under such circumstances, development of a method for detecting a G-quadruplex has been actively attempted using a probe that specifically interacts with a G-quadruplex, and can alter the signal depending on the amount of the G-quadruplex.
For example, PTL 1 proposes a novel compound that is specific for a G-quadruplex. Certainly, this probe is highly selective for binding to a G-quadruplex as compared with single stranded and double stranded DNAs; but binding to the single stranded and double stranded DNAs can be also identified.
In addition, NPL 1 describes specificity for a G-quadruplex of a cationic porphyrin having a structure represented by the following chemical formula 1. In NPL 1, a phenomenon of SPR (surface plasmon resonance) is utilized to analyze binding on a gold substrate the porphyrin with a DNA having a double stranded structure, or with a G-quadruplex. As a result, it is sure that the alteration of the signal is greater with the G-quadruplex compared to with the double stranded DNA, but the alteration of the signal is also observed with the double stranded DNA, thereby leading to failure in observation with sufficient specificity.

Furthermore, NPL 2 reports specificity of a cationic porphyrin having a structure represented by the following Chemical formula 2 or Chemical formula 3 for a G-quadruplex. Also in this NPL 2, analyses of a SPR phenomenon or a ultraviolet visible absorption spectrum are carried out to determine specific binding of these porphyrins to a G-quadruplex. However, it is described that double stranded DNAs and these porphyrins may nonspecifically bound under conditions of the DNA concentration being no lower than 5 μM.

As described above, the probes developed heretofore in attempts to detect a G-quadruplex are all cationic. This would be based on the idea that cationic probes can be very advantageous taking into consideration of the G-quadruplex which is anionic. That is, an anionic probe will cause electrostatic repulsion, which can be disadvantageous in binding reactions. Since not only a G-quadruplex but all DNA strands are anionic, for the development of probes targeting to the same, the most important design guide will be directed to provide cationic one, leading to preclusion of an idea to provide anionic one.